14 research outputs found
NMR resonance assignments of RNase P protein from \u3cem\u3eThermotoga maritima\u3c/em\u3e
Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5ā² precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function
Ī³-Hydroxy-1,N2-propano-2ā²-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage
Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7āAminomethyl-7-deaza-2ā²-deoxyguanosine
A cationic
7-aminomethyl-7-deaza-2ā²-deoxyguanosine (7amG)
was incorporated site-specifically into the self-complementary duplex
dĀ(G<sup>1</sup>A<sup>2</sup>ĀG<sup>3</sup>ĀA<sup>4</sup>Ā<u>X</u><sup>5</sup>ĀC<sup>6</sup>ĀG<sup>7</sup>ĀC<sup>8</sup>ĀT<sup>9</sup>ĀC<sup>10</sup>ĀT<sup>11</sup>C<sup>12</sup>)<sub>2</sub> (<u>X</u> = 7amG). This construct placed two positively charged amines adjacent
to the major groove edges of two symmetry-related guanines, providing
a model for probing how cation binding in the major groove modulates
the structure and stability of DNA. Molecular dynamics calculations
restrained by nuclear magnetic resonance (NMR) data revealed that
the tethered cationic amines were in plane with the modified base
pairs. The tethered amines did not form salt bridges to the phosphodiester
backbone. There was also no indication of the amines being capable
of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function
of temperature revealed that the X<sup>5</sup> imino resonance remained
sharp at 55 Ā°C. Additionally, two 5ā²-neighboring base
pairs, A<sup>4</sup>:T<sup>9</sup> and G<sup>3</sup>:C<sup>10</sup>, were stabilized with respect to the exchange of their imino protons
with solvent. The equilibrium constant for base pair opening at the
A<sup>4</sup>:T<sup>9</sup> base pair determined by magnetization
transfer from water in the absence and presence of added ammonia base
catalyst decreased for the modified duplex compared to that of the
A<sup>4</sup>:T<sup>9</sup> base pair in the unmodified duplex, which
confirmed that the overall fraction of the A<sup>4</sup>:T<sup>9</sup> base pair in the open state of the modified duplex decreased. This
was also observed for the G<sup>3</sup>:C<sup>10</sup> base pair,
where Ī±<i>K</i><sub>op</sub> for the G<sup>3</sup>:C<sup>10</sup> base pair in the modified duplex was 3.0 Ć 10<sup>6</sup> versus 4.1 Ć 10<sup>6</sup> for the same base pair in
the unmodified duplex. In contrast, equilibrium constants for base
pair opening at the X<sup>5</sup>:C<sup>8</sup> and C<sup>6</sup>:G<sup>7</sup> base pairs did not change at 15 Ā°C. These results argue
against the notion that electrostatic interactions with DNA are entirely
entropic and suggest that major groove cations can stabilize DNA via
enthalpic contributions to the free energy of duplex formation
Observation of Two Modes of Inhibition of Human Microsomal Prostaglandin E Synthase 1 by the Cyclopentenone 15-Deoxy-Ī<sup>12,14</sup>-prostaglandin J<sub>2</sub>
Microsomal prostaglandin E synthase 1 (MPGES1) is an
enzyme that
produces the pro-inflammatory molecule prostaglandin E<sub>2</sub> (PGE<sub>2</sub>). Effective inhibitors of MPGES1 are of considerable
pharmacological interest for the selective control of pain, fever,
and inflammation. The isoprostane, 15-deoxy-Ī<sup>12,14</sup>-prostaglandin J<sub>2</sub> (15d-PGJ<sub>2</sub>), a naturally occurring
degradation product of prostaglandin D<sub>2</sub>, is known to have
anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ<sub>2</sub> can inhibit MPGES1 by covalent modification of residue C59
and by noncovalent inhibition through binding at the substrate (PGH<sub>2</sub>) binding site. The mechanism of inhibition is dissected by
analysis of the native enzyme and the MPGES1 C59A mutant in the presence
of glutathione (GSH) and glutathione sulfonate. The location of inhibitor
adduction and noncovalent binding was determined by triple mass spectrometry
sequencing and with backbone amide H/D exchange mass spectrometry.
The kinetics, regiochemistry, and stereochemistry of the spontaneous
reaction of GSH with 15d-PGJ<sub>2</sub> were determined. The question
of whether the anti-inflammatory properties of 15d-PGJ<sub>2</sub> are due to inhibition of MPGES1 is discussed
Unraveling the Aflatoxin-FAPY Conundrum: Structural Basis for Differential Replicative Processing of Isomeric Forms of the Formamidopyrimidine-Type DNA Adduct of Aflatoxin B1
Structural Perturbations Induced by the Ī±-Anomer of the Aflatoxin B<sub>1</sub> Formamidopyrimidine Adduct in Duplex and Single-Strand DNA
The guanine N7 adduct of aflatoxin B<sub>1</sub> <i>exo</i>-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between Ī± and Ī² anomers. The Ī² anomer is highly mutagenic in <i>Escherichia coli</i>, producing G ā T transversions; it thermally stabilizes the DNA duplex. The AFB-Ī±-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-Ī±-FAPY adduct has been elucidated in 5ā²-d(C<sup>1</sup>T<sup>2</sup>A<sup>3</sup>T<sup>4</sup><u>X</u><sup>5</sup>A<sup>6</sup>T<sup>7</sup>T<sup>8</sup>C<sup>9</sup>A<sup>10</sup>)-3ā²Ā·5ā²-d(T<sup>11</sup>G<sup>12</sup>A<sup>13</sup>A<sup>14</sup>T<sup>15</sup>C<sup>16</sup>A<sup>17</sup>T<sup>18</sup>A<sup>19</sup>G<sup>20</sup>)-3ā² (X = AFB-Ī±-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5ā² face of the pyrimidine moiety at the damaged nucleotide between base pairs T<sup>4</sup>Ā·A<sup>17</sup> and X<sup>5</sup>Ā·C<sup>16</sup>, placing the FAPY C5ā<i>N</i><sup>5</sup> bond in the <i>R</i><sub><i>a</i></sub> axial conformation. Large perturbations of the Īµ and Ī¶ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5ā² neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3ā²-neighbor adenine stabilizes the <i>E</i> conformation of the formamide moiety. NMR studies reveal a similar 5ā²-intercalation of the AFB moiety for the AFB-Ī±-FAPY adduct in the tetramer 5ā²-d(C<sup>1</sup>T<sup>2</sup><u>X</u><sup>3</sup>A<sup>4</sup>)-3ā², involving the <i>R</i><sub><i>a</i></sub> axial conformation of the FAPY C5ā<i>N</i><sup>5</sup> bond and the <i>E</i> conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-Ī²-FAPY adduct also intercalates on the 5ā² side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5ā²-stacking leads to the <i>R</i><sub><i>a</i></sub> conformational preference about the C5ā<i>N</i><sup>5</sup> bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-Ī±-FAPY adduct explain its lower stability as compared to the AFB-Ī²-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3ā²-neighboring adenine stabilizing the <i>E</i> configuration of the formamide moiety is also observed for the AFB-Ī²-FAPY adduct, and suggests that the identity of the 3ā²-neighbor nucleotide modulates the stability and biological processing of AFB adducts
Correction to Differential Stabilities and Sequence-Dependent Base Pair Opening Dynamics of WatsonāCrick Base Pairs with 5-Hydroxymethylcytosine, 5-Formylcytosine, or 5-Carboxylcytosine
Ī³-Hydroxy-1,<i>N</i><sup><i>2</i></sup>-propano-2ā²-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage
The Ī³-hydroxy-1,<i>N</i><sup><i>2</i></sup>-propano-2ā²-deoxyguanosine adduct (Ī³-OH-PdG) was introduced into 5ā²-d(GCTAGC<u>X</u>AGTCC)-3ā²Ā·5ā²-d(GGACTCGCTAGC)-3ā² (<u>X</u> = Ī³-OH-PdG). In the presence of excess peptide KWKK, <sup>13</sup>C isotope-edited NMR revealed the formation of two spectroscopically distinct DNAāKWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the <i>N</i><sup><i>2</i></sup>-dG propylaldehyde tautomer of the Ī³-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited <sup>15</sup>N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH<sub>3</sub>, suggesting that they existed, in part, as imine (Schiff base) linkages. However, <sup>13</sup>C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNAāKWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. (2010) Biochemistry, 49, 6155ā6164]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the <i>S</i>-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNAāKWKK conjugates thermally stabilized duplex DNA. However, the DNAāKWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5ā²-CpX-3ā² sequence, the DNAāKWKK conjugates slowly converted to interstrand <i>N</i><sup>2</sup>-dG:<i>N</i><sup>2</sup>-dG DNA cross-links and ring-opened Ī³-OH-PdG derivatives over a period of weeks